1. Field of the Invention
The invention pertains to the field of virion production. More specifically, the invention pertains to methods for increasing the titer of retroviral vectors.
2. Description of Related Art
Viruses are very efficient at infecting cells and causing them to produce new viral particles. Highly infective viral vectors can be designed that cause the cell to manufacture a desired product. Retroviruses, a family of viruses, are especially efficient at this. Vesicular Stomatitis Virus (VSV), a rhabdovirus, utilizes its envelope glycoprotein (G protein) for viral entry and viral particle production.
Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) is a well-studied insect baculovirus that promiscuously enters a variety of human and animal cells. AcMNPV requires its envelope glycoprotein, GP64, for viral entry and efficient viral particle production.
Baculoviruses are large double stranded DNA viruses that have been studied as agents for biological control of insect pests, as expression vectors for high level production of heterologous proteins, and as transduction vectors and potential agents for human gene therapy. The latter applications derive from the observation that baculovirus virions can efficiently enter a variety of human and other animal cell types and deliver the baculovirus DNA genome to the nucleus of the cell. Viral entry is relatively efficient and promiscuous, permitting entry into many different cell types that are not permissive for viral replication. Expression of foreign proteins in heterologous (non-permissive) cells is achieved by engineering the coding sequence of a foreign gene under a promoter that is active in the target cell type. For example, protein expression in human cells is achieved by placing the coding region of the foreign gene under the control of a promoter that is active in human cells, e.g. a human cytomegalovirus (HCMV) early promoter. Attempts to expand the range of cells that are promiscuously entered by baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) virions include studies in which the human Vesicular Stomatitis Virus envelope glycoprotein known as G (VSV G) was expressed in addition to the baculovirus envelope protein GP64. For many applications in gene therapy however, targeted entry of the baculovirus virion into specific cell types would be highly desirable in order to either positively modify or negatively affect the growth and/or survival of the target cell type. For example, cells infected with viruses such as HIV might be targeted for destruction or death. Alternatively, genetic defects might be corrected by expression of a protein in specific cell types. Thus, the ability to target baculovirus virion entry to specific cell types would be of great value as a biotechnological tool in medicine or gene therapy. However, because of the promiscuity of AcMNPV entry into heterologous cells, targeted entry is not currently possible with native virions.
AcMNPV is the baculovirus studied most extensively for gene therapy applications. AcMNPV requires the major envelope glycoprotein known as GP64 for virion production and viral entry. In the absence of GP64, baculovirus virion production is severely reduced and the virions that are produced are not infectious. Thus, GP64 is critical for the efficient production of virions and for the ability of those virions to enter host cells. Similarly, other baculoviruses such as LdMNPV, SeMNPV, or HaNPV require the so-called F envelope protein for entry. Studies have shown that virions of the baculovirus AcMNPV can be pseudotyped—that is, the GP64 protein can be replaced with the envelope protein from another virus. AcMNPV viruses lacking GP64 (gp64null) that express the human Vesicular Stomatitis Virus G (VSV G) protein are able to produce infectious virions. However, like GP64, entry mediated by the VSV G protein is known to be highly promiscuous. Thus, VSV G does not provide specificity in cell targeting. Studies pseudotyping AcMNPV with F envelope proteins from other baculoviruses showed that some but not all baculovirus F proteins could substitute for GP64. In addition, studies of baculovirus F proteins in pseudotyped retroviruses indicate that they may not be useful in gene therapy applications as they did not mediate efficient entry into mammalian (mouse) cells.